Colon cancer is the fourth leading cause of cancer mortality in men and women. The 5-year survival rate is 61% over all stages with early detection being a prerequisite for curative therapy of the disease. Up to 95% of all colorectal cancers are adenocarcinomas of varying differentiation grades.
Sporadic colon cancer develops in a multistep process starting with the pathological transformation of normal colonic epithelium to an adenoma which consecutively progresses to invasive cancer. The progression rate of colonic adenomas is currently predicted based on their histological appearance, location, degree of spread and extent of bowel involvement. For example, tubular-type benign adenomas rarely progress to malignant tumours, whereas villous benign adenomas, particularly if larger than 2 cm in diameter, have a significant malignant potential.
During progression from benign proliferative lesions to malignant neoplasms several genetic and epigenetic alterations are known to occur. Somatic mutation of the APC gene seems to be one of the earliest events in 75 to 80% of colorectal adenomas and carcinomas. Activation of K-RAS is thought to be a critical step in the progression towards a malignant phenotype. Consecutively, mutations in other oncogenes as well as alterations leading to inactivation of tumor suppressor genes accumulate.
Aberrant DNA methylation within CpG islands is among the earliest and most common alterations in human cancers leading to abrogation or overexpression of a broad spectrum of genes. In addition, abnormal methylation has been shown to occur in CpG rich regulatory elements in intronic and coding parts of genes for certain tumours. In contrast to the specific hypermethylation of tumour suppressor genes, an overall hypomethylation of DNA can be observed in tumour cells. This decrease in global methylation can be detected early, far before the development of frank tumour formation. Also, correlation between hypomethylation and increased gene expression was reported for many oncogenes. In colon cancer, aberrant DNA methylation constitutes one of the most prominent alterations and inactivates many tumour suppressor genes such as p14ARF, p16INK4a, THBS1, MINT2, and MINT31 and DNA mismatch repair genes such as hMLH1.
In the molecular evolution of colorectal cancer, DNA methylation errors have been suggested to play two distinct roles. In normal colonic mucosa cells, methylation errors accumulate as a function of age or as time-dependent events predisposing these cells to neoplastic transformation. For example, hypermethylation of several loci could be shown to be already present in adenomas, particularly in the tubulovillous and villous subtype. At later stages, increased DNA methylation of CpG islands plays an important role in a subset of tumours affected by the so called CpG island methylator phenotype (CIMP). Most CIMP+ tumours, which constitute about 15% of all sporadic colorectal cancers, are characterized by micro satellite instability (MIN) due to hypermethylation of the hMLH1 promoter and other DNA mismatch repair genes. By contrast, CIMP− colon cancers evolve along a more classic genetic instability pathway (CIN), with a high rate of p53 mutations and chromosomal changes.
However, the molecular subtypes do not only show varying frequencies regarding molecular alterations. According to the presence of either micro satellite instability or chromosomal aberrations, colon cancer can be subclassified into two classes, which also exhibit significant clinical differences. Almost all MIN tumours originate in the proximal colon (ascending and transversum), whereas 70% of CIN tumours are located in the distal colon and rectum. This has been attributed to the varying prevalence of different carcinogens in different sections of the colon. Methylating carcinogens, which constitute the prevailing carcinogen in the proximal colon have been suggested to play a role in the pathogenesis of MIN cancers, whereas CIN tumours are thought to be more frequently caused by adduct-forming carcinogens, which occur more frequently in distal parts of the colon and rectum. Moreover, MIN tumours have a better prognosis than do tumours with a CIN phenotype and respond better to adjuvant chemotherapy.
The identification of markers for the differentiation of colon carcinoma as well as for early detection are main goals of current research.
The alpha-calcitonin gene encodes a small family of peptides: calcitonin, katacalcin, and calcitonin gene-related peptide (CGRP). Calcitonin is concerned with skeletal integrity, the secretion of calcitonin is, in part, oestrogen dependent, and it appears likely that a postmenopausal decline in calcitonin secretion is a factor in the development of postmenopausal osteoporosis.
Investigation of the Calcitonin gene has revealed that hypermethylation of the promoter region of the gene is present in neoplastic cells of several cancer types, particularly acute leukemias. The major part of said research was carried out using methylation sensitive enzyme based methods, this identified the general phenomenon of hypermethylation within the promoter and first exon regions of the gene in multiple types of cancers. However said methods do not allow for a targeted analysis of selected CpG positions. The observations of hypermethylation were enabled only by the assumption of comethylation within the region. Comethylation is a phenomenon whereby methylation of one CpG position is taken as indicative of methylation of all CpG positions within the region. Examples of research carried out using restriction enzyme based methods include the following:    Hiltunen M O, Koistinaho J, Alhonen L, Myohanen S, Marin S, Kosma V M, Paakkonen M, Janne J. Hypermethylation of the WT1 and calcitonin gene promoter regions at chromosome 11p in human colorectal cancer. Br J Cancer. 1997; 76(9):1124-30.    Silverman A L, Park J G, Hamilton S R, Gazdar A F, Luk G D, Baylin S B. Abnormal methylation of the calcitonin gene in human colonic neoplasms. Cancer Res. 1989 Jul. 1; 49(13):3468-73.
The gene “Versican,” (NM_004385) also known as CSPG2 encodes a large chondroitin sulfate proteoglycan. This gene is known to exhibit aberrant methylation patterns and conflicting opinions on this matter have been published. For instance Adany R and Iozzo R V. (“Altered methylation of versican proteoglycan gene in human colon carcinoma.” Biochem Biophys Res Commun 1990 Sep. 28; 171(3):1402-13) observed a correlation between hypomethylation and colonic neoplasms. However, more recently Issa et. al. (“Accelerated Age-related CpG Island Methylation in Ulcerative Colitis,” Cancer Research 61, 3573-3577, May 1, 2001) described an observed hypermethylation of dysplastic mucosa as compared to non-UC control mucosa (58% versus 31%, P=0.01) or compared with adjacent uninvolved mucosa (58% versus 35%, P=0.06). Therefore it would seem that although aberrant methylation of this gene has been observed in colorectal cell proliferative disorders, the characterisation of this aberrant methylation is as yet not obvious and it would appear that the commonly held assumption of co-methylation does not hold in the case of this gene.
The gene TPEF (also known as TMEFF2) NM_016192 encodes a transmembrane protein containing EGF and follistatin domains. It was initially identified on the basis of its methylation properties by Jones et. al. (“The Gene for a Novel Transmembrane Protein Containing Epidermal Growth Factor and Follistatin Domains Is Frequently Hypermethylated in Human Tumor Cells,” Cancer Research 60, 4907-4912, Sep. 1, 2000). It was therein shown that the 5′ region of the gene contained a CpG island, a 3′ region of which was shown to exhibit significant hypermethylation in tumor cell lines. Although significant said observation was carried out by means of arbitrarily primed PCR, a methodology that is not suitable for application in a clinical or diagnostic setting.
EYA4 is the most recently identified member of the vertebrate Eya (eyes-absent) gene family, a group of four transcriptional activators that interact with other proteins in a conserved regulatory hierarchy to ensure normal embryologic development. The EYA4 gene is mapped to 6q22.3 and encodes a 640 amino acid protein. The structure of EYA4 conforms to the basic pattern established by EYA1-3, and includes a highly conserved 271 amino acid C-terminus called the eya-homologous region (eyaHR; alternatively referred to as the eya domain or eya homology domain 1) and a more divergent proline-serine-threonine (PST)-rich (34-41%) transactivation domain at the N-terminus. EYA proteins interact with members of the SIX and DACH protein families during early embryonic development. Mutations in the EYA4 gene are responsible for postlingual, progressive, autosomal dominant hearing loss at the DFNA10 locus (Wayne S, Robertson N G, DeClau F, Chen N, Verhoeven K, Prasad S, Tranebjarg L, Morton C C, Ryan A F, Van Camp G, Smith R J: Mutations in the transcriptional activator EYA4 cause late-onset deafness at the DFNA10 locus. Hum Mol Genet 2001 Feb. 1; 10(3):195-200 with further references). A link between the Methylation of Cytosine positions in the EYA 4 gene and cancer has not yet been established.
The cadherins are a family of cell surface glycoproteins responsible for selective cell recognition and adhesion. Several family members, including CDH1 (E-cadherin) and CDH13 (H-cadherin, NM_001257) are located on the long arm of chromosome 16, while another gene cluster resides on the short arm of chromosome 5. The chromosomal locations of several of the cadherins are sites of frequent loss of heterozygosity in many tumor types. Deletions of 16q are frequent in breast, lung, and other carcinomas. Loss of expression of cadherins has been described in many epithelial cancers, and it may play a role in tumour cell invasion and metastasis CDH13 expression is diminished in breast and lung cancers. In ovarian tumours, the combination of deletion and aberrant methylation has been reported to inactivate CDH13. Aberrant methylation of CDH13 has also been reported in lung cancers.
5-methylcytosine is the most frequent covalent base modification in the DNA of eukaryotic cells. It plays a role, for example, in the regulation of the transcription, in genetic imprinting, and in tumorigenesis. Therefore, the identification of 5-methylcytosine as a component of genetic information is of considerable interest. However, 5-methylcytosine positions cannot be identified by sequencing since 5-methylcytosine has the same base pairing behaviour as cytosine. Moreover, the epigenetic information carried by 5-methylcytosine is completely lost during PCR amplification.
A relatively new and currently the most frequently used method for analysing DNA for 5-methylcytosine is based upon the specific reaction of bisulfite with cytosine which, upon subsequent alkaline hydrolysis, is converted to uracil which corresponds to thymidine in its base pairing behaviour. However, 5-methylcytosine remains unmodified under these conditions. Consequently, the original DNA is converted in such a manner that methylcytosine, which originally could not be distinguished from cytosine by its hybridisation behaviour, can now be detected as the only remaining cytosine using “normal” molecular biological techniques, for example, by amplification and hybridisation or sequencing. All of these techniques are based on base pairing which can now be fully exploited. In terms of sensitivity, the prior art is defined by a method which encloses the DNA to be analysed in an agarose matrix, thus preventing the diffusion and renaturation of the DNA (bisulfite only reacts with single-stranded DNA), and which replaces all precipitation and purification steps with fast dialysis (Olek A, Oswald J, Walter J. A modified and improved method for bisulphite based cytosine methylation analysis. Nucleic Acids Res. 1996 Dec. 15; 24(24):5064-6). Using this method, it is possible to analyse individual cells, which illustrates the potential of the method. However, currently only individual regions of a length of up to approximately 3000 base pairs are analysed, a global analysis of cells for thousands of possible methylation events is not possible. However, this method cannot reliably analyse very small fragments from small sample quantities either. These are lost through the matrix in spite of the diffusion protection.
An overview of the further known methods of detecting 5-methylcytosine may be gathered from the following review article: Rein, T., DePamphilis, M. L., Zorbas, H., Nucleic Acids Res. 1998, 26, 2255.
To date, barring few exceptions (e.g., Zeschnigk M, Lich C, Buiting K, Doerfler W, Horsthemke B. A single-tube PCR test for the diagnosis of Angelman and Prader-Willi syndrome based on allelic methylation differences at the SNRPN locus. Eur J Hum Genet. 1997 March-April; 5(2):94-8) the bisulfite technique is only used in research. Always, however, short, specific fragments of a known gene are amplified subsequent to a bisulfite treatment and either completely sequenced (Olek A, Walter J. The pre-implantation ontogeny of the H19 methylation imprint. Nat Genet. 1997 November; 17(3):275-6) or individual cytosine positions are detected by a primer extension reaction (Gonzalgo M L, Jones P A. Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res. 1997 Jun. 15; 25(12):2529-31, WO 95/00669) or by enzymatic digestion (Xiong Z, Laird P W. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 1997 Jun. 15; 25(12):2532-4). In addition, detection by hybridization has also been described (Olek et al., WO 99/28498).
Further publications dealing with the use of the bisulfite technique for methylation detection in individual genes are: Grigg G, Clark S. Sequencing 5-methylcytosine residues in genomic DNA. Bioessays. 1994 June; 16(6):431-6, 431; Zeschnigk M, Schmitz B, Dittrich B, Buiting K, Horsthemke B, Doerfler W. Imprinted segments in the human genome: different DNA methylation patterns in the Prader-Willi/Angelman syndrome region as determined by the genomic sequencing method. Hum Mol Genet. 1997 March; 6(3):387-95; Feil R, Charlton J, Bird A P, Walter J, Reik W. Methylation analysis on individual chromosomes: improved protocol for bisulphite genomic sequencing. Nucleic Acids Res. 1994 Feb. 25; 22(4):695-6; Martin V, Ribieras S, Song-Wang X, Rio M C, Dante R. Genomic sequencing indicates a correlation between DNA hypomethylation in the 5′ region of the pS2 gene and its expression in human breast cancer cell lines. Gene. 1995 May 19; 157(1-2):261-4; WO 97/46705 and WO 95/15373.
An overview of the Prior Art in oligomer array manufacturing can be gathered from a special edition of Nature Genetics (Nature Genetics Supplement, Volume 21, January 1999), published in January 1999, and from the literature cited therein.
Fluorescently labeled probes are often used for the scanning of immobilized DNA arrays. The simple attachment of Cy3 and Cy5 dyes to the 5′-OH of the specific probe are particularly suitable for fluorescence labels. The detection of the fluorescence of the hybridized probes may be carried out, for example via a confocal microscope. Cy3 and Cy5 dyes, besides many others, are commercially available.
Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI-TOF) is a very efficient development for the analysis of biomolecules (Karas M, Hillenkamp F. Laser desorption ionisation of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988 Oct. 15; 60(20):2299-301). An analyte is embedded in a light-absorbing matrix. The matrix is evaporated by a short laser pulse thus transporting the analyte molecule into the vapor phase in an unfragmented manner. The analyte is ionised by collisions with matrix molecules. An applied voltage accelerates the ions into a field-free flight tube. Due to their different masses, the ions are accelerated at different rates. Smaller ions reach the detector sooner than bigger ones.
MALDI-TOF spectrometry is excellently suited to the analysis of peptides and proteins. The analysis of nucleic acids is somewhat more difficult (Gut I G, Beck S. DNA and Matrix Assisted Laser Desorption Ionisation Mass Spectrometry. Current Innovations and Future Trends. 1995, 1; 147-57). The sensitivity to nucleic acids is approximately 100 times worse than to peptides and decreases disproportionally with increasing fragment size. For nucleic acids having a multiply negatively charged backbone, the ionisation process via the matrix is considerably less efficient. In MALDI-TOF spectrometry, the selection of the matrix plays an eminently important role. For the desorption of peptides, several very efficient matrixes have been found which produce a very fine crystallisation. There are now several responsive matrixes for DNA, however, the difference in sensitivity has not been reduced. The difference in sensitivity can be reduced by chemically modifying the DNA in such a manner that it becomes more similar to a peptide. Phosphorothioate nucleic acids in which the usual phosphates of the backbone are substituted with thiophosphates can be converted into a charge-neutral DNA using simple alkylation chemistry (Gut I G, Beck S. A procedure for selective DNA alkylation and detection by mass spectrometry. Nucleic Acids Res. 1995 Apr. 25; 23(8):1367-73). The coupling of a charge tag to this modified DNA results in an increase in sensitivity to the same level as that found for peptides. A further advantage of charge tagging is the increased stability of the analysis against impurities which make the detection of unmodified substrates considerably more difficult.
Genomic DNA is obtained from DNA of cell, tissue or other test samples using standard methods. This standard methodology is found in references such as Sambrook, Fritsch and Maniatis eds., Molecular Cloning: A Laboratory Manual, 1989.